A simple, sensitive and rapid liquid-liquid extraction method for the analysis of nicotinic acid (niacin) and its labeled internal standard nicotinic acid-d4 (niacin-d4) in human plasma was developed and validated. The analyte and its internal standard were isolated from acidified plasma using a single liquid-liquid extraction procedure with methyl-t-butyl ether. The extracted samples were analyzed by liquid chromatography-tandem mass spectrometry in positive electrospray ionization mode with multiple reaction monitoring. The calibration curves were linear in the measured range between 5 and 1000 ng/mL and the limit of detection was calculated as 122 pg/mL. The method required 250 microL of human plasma and the total run time between injections was 3.5 min. Matrix effects were assessed by post-column infusion experiments, phospholipids monitoring and post-extraction addition experiments. The extraction of phospholipids and niacin from plasma was studied under acidic, neutral and basic conditions. Acidic conditions were optimal for both the recovery of niacin and the removal of phospholipids; the degree of matrix effects for niacin was determined to be 2.5%. It was concluded that effective removal of matrix components can overcome low recovery issues associated with liquid-liquid extractions of polar analytes.
A miniaturized system based on microfluidic capillaries is presented for point-of-care testing and clinical assessment. The approach relies on microsyringe pump-generated flow to deliver reagents and immunoaffinity chromatography to isolate the antigen from biological matrixes. Capillary sandwich immunoassays for C-reactive protein (CRP) were demonstrated in human serum and cerebrospinal fluid (CSF), which are relevant matrixes for cardiovascular disease risk and meningitis research, respectively. Capillaries packed with antibody-coated silica beads were used to capture CRP from the matrix and a second, dye-labeled antibody was introduced to form a sandwich complex. An acidic elution buffer dissociated the antibody-antigen complexes, and the labeled antibody was detected with diode laser-induced fluorescence. Four parameter logistic functions and % relative error plots were used to model and assess the data. The calibration ranges for CRP were 0.05-3.0 microg/mL in 1:10 diluted serum and 0.01-30 microg/mL in undiluted CSF. The microfluidic apparatus employed a flow rate of 2 microL/min and a sample injection volume of 250 nL. Since it was not necessary to reach antibody-antigen reaction equilibrium and the assay platform dimensions were minimal, run times were as short as 10 min.
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